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Engineering of a TrpR-Based Biosensor for Altered Dynamic Range and Ligand Preference.

Xinyu Gong1, Ruihua Zhang1, Jian Wang1

  • 1School of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, 302 East Campus Road, Athens, Georgia 30602, United States.

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Summary

Engineered tryptophan repressor (TrpR) biosensors in E. coli show improved dynamic range and ligand specificity. These enhanced biosensors are valuable for high-throughput screening and dynamic regulation in biosynthesis.

Keywords:
5-hydroxytryptophanTrpRbiosensordynamic behaviortryptophan

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Transcriptional factors regulate cellular functions, and their dynamic behavior is key for biosensor applications.
  • TrpR-based tryptophan repressor systems are crucial for understanding and manipulating cellular processes.
  • Optimizing biosensor dynamic range and ligand specificity is essential for advanced applications.

Purpose of the Study:

  • To characterize and engineer a TrpR-based tryptophan repressor system (TrpR1-PtrpO1) in Escherichia coli.
  • To improve the dynamic behavior, ligand specificity, and operational range of the biosensor.
  • To develop enhanced genetic elements for high-throughput screening and dynamic regulation in biosynthesis.

Main Methods:

  • Rational mutation of ligand-binding pocket residues (I57, V58) to alter ligand specificity.
  • Tuning regulator-operator binding affinity by mutating the TrpR1 binding box.
  • Characterization of engineered TrpR1 variants (V58E, V58K) for tryptophan and 5-HTP preference.

Main Results:

  • Engineered TrpR1 variants (V58E, V58K) demonstrated altered ligand preference for tryptophan and 5-HTP.
  • Biosensor-induced expression levels increased up to 10-fold with engineered variants.
  • The TrpR1-PtrpO1 system exhibited improved dynamic range and ligand preference after engineering.

Conclusions:

  • The TrpR1-PtrpO1 biosensor can be effectively engineered for extended dynamic ranges and improved ligand preference.
  • Engineered biosensor variants with enhanced dynamic behavior serve as valuable genetic tools.
  • This work facilitates high-throughput screening and dynamic regulation in biosynthetic scenarios.